The human dopamine transporter is a target of cocaine, clinically relevant drugs used to treat attention deficit disorder, movement disorders, nicotine addiction and probes for imaging dopamine neurons in living brain The broad significance of the dopamine transporter offers a compelling reason to investigate the molecular mechanisms by which the transporter functions and is blocked by clinically relevant drugs We are investigating a hypothesis that aromatic amino acids in the human dopamine transporter are critical for drug binding and dopamine transport This hypothesis is based on recent findings from our laboratory that non-amines, which contain no amine nitrogen, retain high affinity binding and blockade of monoamine transport As non-amines do not participate in ionic or hydrogen bonding to the transporter, the aromatic ring of the drug and transporter may contribute to the formation of a drug-transporter complex This hypothesis is being investigated by site-dire cted mutagenesis of rationally selected aromatic amino acid residues on the human dopamine transporter We express the mutant transporter constructs in the HEK-293 cell line, and investigate their capacity to transport [3H]dopamine and ability to bind [3H]non-amines and their monoamine counterparts Our initial data demonstrates that mutation of specific aromatic amino acid residues to alanine in the dopamine transporter results in profound effects on the formation of drug-transporter complexes, and on dopamine transport The effects of certain mutations can be restored by replacing the mutated alanine residue with a different aromatic residue These studies will contribute to understanding the mechanisms by which therapeutic and addictive drugs block the dopamine transporter They will also provide needed information for our ongoing program of developing novel drugs for the diagnosis and treatment of dopamine-related neuropsychiatric diseases